In light microscopy of three-dimensionally extended samples, also designated below also as volume samples, high-quality three-dimensional imaging is of key interest. One of the challenges here is presented by imaging errors which are caused by the path of the light in the sample. In this case, in particular attention needs to be paid to so-called spherical aberrations, which result from the fact that the index of refraction of the volume sample is different from the index of refraction of the medium surrounding the sample. The longer the path of the light within the sample, the greater the spherical aberration caused by such maladjustment of the index of refraction. As a result it is particularly difficult to precisely image object planes which are located deep inside the volume sample. If the index of refraction of the sample varies along the optical axis of the microscope objective, i.e. in the direction of the sample depth, it becomes even more difficult to achieve high-quality three-dimensional imaging of the sample.
In order to prevent the imaging errors described above, immersion objectives are generally used, which comprise correcting elements which are adjustable for correction of the spherical aberration. Such correcting elements which are known from the prior art have, for example, lens mounts which are adjustable by means of a motor and by which a lens unit contained in the microscope objective can be displaced along the optical axis for correction of the imaging error. An example of such a correcting adjustment is disclosed in DE 10 2011 051 677 A1.
If the spherical aberration caused by a maladjustment of the index of refraction is to be corrected in a wide axial range, it is necessary to provide the correcting element with a specific refractive power. As a result, during the correcting adjustment, an unwanted change to the object focal distance can occur, i.e. a displacement of the imaged object plane along the optically axis. Such a change to the object focal distance then leads to an intolerable distortion of the recorded volume image.
With regard to the prior art, reference is also made to concepts which provide a depth-dependent adjustment of microscopy parameters, for example the laser power in multiphoton microscopes. However, these parameters do not influence the correction of the microscope objective with respect to its imaging performance or its focal distance setting. Thus the current prior art does not allow high-quality microscopic volume imaging which is extensive in the axial direction, in particular in samples which have an inhomogeneous index of refraction above the sample depth.